This application proposes a translational partnership between academia (University of Virginia and Michigan State University), the public health sector (Commonwealth of Virginia Division of Consolidated Laboratory Services), and industry (IDX Labs LLC) to improve molecular diagnosis of food and waterborne enteropathogens. The underlying hypothesis to this work is that nucleic acid detection will remain for the foreseeable future the most practical means for referral public health laboratories to detect the multiplicity of potential pathogens that inhabit the differential diagnosis of a food or waterborne diarrheal outbreak. Three independent but linked products will be developed on pathways towards clinical use. Aim 1, Dr. Houpt of UVA will expand existing work on protozoa multiplex PCR with Luminex bead-based amplicon detection to include the additional Category B protozoa, bacteria, and viruses. The goal product are three bead-PCR panels that can be used within the existing public health lab triage system of acute, mid, and late-incubation diarrheal disease. Aim 2, Dr. Young of MSU will expand his work on T-RFLP for bacterial 16S rRNA communities in stool to the protozoa (18S rRNA) and then generate a database of T-RFLP patterns from known-diagnosis and no-diagnosis stool specimens to determine whether T-RFLP profiling can provide a diagnostic pigeonhole in the inevitable >50% of diarrheal cases where no pathogen is identified. Aim 3, Dr. Icenhour of IDX Labs will develop oligo-targeted DNA/RNA extraction procedures amidst the complex mixture of stool to increase sensitivity towards the goal of culture (100 CPU bacteria/g). Extraction procedures will be designed for the real-world scenario, adapting them to high-throughput robotics and evaluating utility in the widely-used Cary-Blair media and PVA and formalin fixatives. Aim 4, Dr. Toney of Virginia DCLS and Dr. Maro of Kilimanjaro Christian Medical Center, Tanzania will test these tools in the field in parallel with gold-standard assays in order to guide development. These two sites provide superb access to Category B bacteria and protozoa, respectively. The overall goal is to develop sensitive PCR-based diagnostics directly usable on stool with comparable or better sensitivity to existing technologies, which have clear deficincies in sensitivity of pathogen detection, throughput, and specimen handling.